Detection of neurodegenerative disorders

ABSTRACT

The present invention relates to methods of detecting neurodegenerative diseases or disorders, particularly to methods for early detection of neurodegenerative diseases, such as Parkinson&#39;s disease. The present invention provides a method for detecting a neurodegenerative disease in a subject, the method comprising testing the subject for an indicator of release of neuromelanin from cells in the brain, wherein a positive test is indicative of death of brain cells containing neuromelanin and is characterised by an elevated level of the indicator of release of neuromelanin compared to control values.

TECHNICAL FIELD

The present invention is directed generally to methods of detectingneurodegenerative diseases or disorders, particularly to methods forearly detection of Parkinson's disease.

BACKGROUND ART

Parkinson's disease (PD) is a progressive neurodegenerative diseasecharacterised by motor dysfunction. A clinical diagnosis is made on thebasis of a triad of motor symptoms: tremor, rigidity and bradykinesia orslowness of movement. Pathologically, PD is characterised by the deathof the neurons in an area of the brain called the substantia nigra (SN)which produce the neurotransmitter dopamine (DA).

A major focus of current research is to identify strategies by which thedeath of these neurons can be slowed or halted, thus curbing theprogression of the disease. It is likely that such strategies, termed“neuroprotection”, will be maximally efficacious when the proportionalloss of dopaminergic neurons is relatively low. Post-mortem (Fearnleyand Lees, 1991) and imaging studies (Morrish et al. 1998) suggest,however, that the rate of loss of dopaminergic neurons throughout thedisease course is not linear but decelerates exponentially. Dopaminergicneuron death occurs rapidly during the first 7 years of the diseaseresulting in the loss of at least 65% of total SN dopaminergic neuronnumber during this period, although average neuron loss is much greater(Halliday et al., 1996). As the brain has a substantial capacity forcompensation no motor symptoms are evident at this time. Thus, thisperiod of rapid neuron loss in the absence of clinical signs is termed“preclinical disease”. Neurological signs, and thus a clinicaldiagnosis, occurs only after at least 65% of the neurons are lost, thusthe majority of neurodegeneration in this disease occurs before theclinical onset and diagnosis of the disease.

The rate of loss of the remaining 35% of the neurons proceeds at asignificantly slower rate during the remaining 10 to 20 years of thedisease, the so-called “clinical phase” (FIG. 1). The pattern of cellloss presents a challenge in that the time of the most rapid cell loss,which represents the optimal time-point for the initiation ofneuroprotective strategies, occurs in the absence of an identifiableclinical syndrome.

Accordingly, there is a need to develop a means for detecting aneurodegenerative disease in a subject, and preferably a means fordetecting a neurodegenerative disease during this “preclinical” phase.

The present invention involves the detection of an indicator of therelease of a substance called neuromelanin (NM) from the cytoplasm ofthe dying cells. NM is a complex polymer pigment believed to be formedfrom oxidized dopamine products within the dopaminergic neurons of theSN (and the noradrenergic neurons of the locus ceruleus (LC)). NMusually occurs as granules which can be seen in the cell body but as aconsequence of cell death NM is released into the extracellular space(FIG. 2).

Therefore, the present invention provides a method for detecting aneurodegenerative disease in a subject, the method comprising testingthe subject for an indicator of release of neuromelanin from cells inthe brain, wherein a positive test is indicative of death of brain cellscontaining neuromelanin and is characterised by an elevated level of theindicator of release of neuromelanin compared to control values. Theidentification of a specific marker for the death of these melanisedcells provides a means for detecting the disorders characterised by thedeath of these cells, even prior to the onset of clinical symptoms.

SUMMARY OF INVENTION

In a first aspect, the present invention provides a method for detectinga neurodegenerative disease in a subject, the method comprising testingthe subject for an indicator of release of neuromelanin from cells inthe brain, wherein a positive test is indicative of death of brain cellscontaining neuromelanin and is characterised by an elevated level of theindicator of release of neuromelanin compared to control values.

In a second aspect of the invention, there is provided a method todetect a neurodegenerative disease in a subject who is tested prior tohaving any clinical symptoms of said neurodegenerative disease, whereinsaid method comprises testing the subject for an indicator of release ofneuromelanin from cells in the brain, wherein a positive test isindicative of death of brain cells containing neuromelanin and ischaracterised by an elevated level of the indicator of release ofneuromelanin compared to control values.

Typically, cell death is associated with the neurodegenerative diseasein the subject.

Typically, the clinical symptoms of the neurodegenerative disease arethe classical symptoms of such a disease. More typically, these symptomsinclude tremor, rigidity and bradykinesia or slowness of movement.

Preferably, the indicator is an immune response in the form ofcirculating antibodies to neuromelanin (NM), or analogues thereof, inthe subject.

In a preferred form, the method according to the first or second aspectof the present invention employs the detection of antibodies capable ofreacting to NM, or an antigenic fragment or analogue thereof, present ina subject.

Preferably, the analogue of NM is selected from the group consisting ofsynthetic dopamine melanin and synthetic noradrenaline melanin.

In a preferred form, the method of the first or second aspect comprises:

-   -   (i) obtaining a blood sample from said subject;    -   (ii) isolating sera from said blood;    -   (iii) incubating said sera with an antigen selected from human        NM or synthetic dopamine melanin under conditions suitable for        antibody-antigen binding; and    -   (iv) detecting bound antibody.

Typically, detection of bound antibody utilises labelled anti-human IgG.

In a third aspect, the present invention provides a method of treatmentof a neurodegenerative disease in a subject, the method comprising:

-   -   (a) screening subjects to detect a neurodegenerative disease        using the method according to the first or second aspect of the        present invention; and    -   (b) initiating treatment for subjects tested positive for the        indicator of release of neuromelanin from cells in the brain.

Typically, the treatment includes, but is not limited to, administeringa therapeutically effective amount of at least one of the following:antioxidants, iron chelators, nonamine oxidase inhibitors, apoptosisinhibitors, growth factors, dopamine receptor inhibitors, endogenousenzymes which protect against oxidative damage such as glutathione,superoxide dismutase and catalase, inhibitors of excitatory damage,zonisamide, benzamide compounds, or ethanesulfonyl-piperidinederivatives, or a combination thereof.

In a fourth aspect, the present invention provides a system for thedetection of a neurodegenerative disease in a subject, the systemcomprising:

-   (a) means for capturing an indicator of release of neuromelanin from    cells in the brain of a subject; and-   (b) means for detecting the captured indicator of release of    neuromelanin from cells in the brain.

When the indicator of release of neuromelanin is circulating antibodiesin a subject, the means for capturing the indicator of release of NM ispreferably neuromelanin or an antigenic fragment or analogue thereofimmobilized to a solid surface. In this situation, a detectably labelledprobe is applied which is specific for antibodies captured on the solidsupport via the neuromelanin, fragment or analogue. The means fordetecting the captured indicator can be any suitable means for detectingthe presence of the label on the probe.

In a preferred form, the system according to the fourth aspect of thepresent invention is a kit comprising neuromelanin or an antigenicfragment or an analogue thereof bound to a solid support and a source ofa detectably labelled probe, wherein, in use, a subject's serum sampleis applied to the solid support such that any antibodies to neuromelaninwill bind to the support via the neuromelanin or an antigenic fragmentor analogue thereof, a sample of the probe is added and allowed to bindto the bound antibody, and the label on the bound probe is detected.

Typically, the probe is a labelled antihuman IgG.

More typically, the probe is horseradish peroxidase-conjugated goatantihuman IgG.

Typically, the components of the kit are housed in separate containers.

Typically, the kit may further comprise one or more other containers,containing other components, such as wash reagents, and other reagentscapable of detecting the presence of bound antibodies. More typically,the detection reagents may include reagents capable of reacting with thelabelled probe.

An ELISA-based system is particularly suitable for this aspect of theinvention.

In a fifth aspect, the present invention relates to the use of thesystem according to the fourth aspect of the present invention to detecta neurodegenerative disease in a subject.

Typically, a method for detecting a neurodegenerative disease in asubject using the system according to the fourth aspect of the presentinvention comprises testing the subject for an indicator of release ofneuromelanin from cells in the brain, the method comprising

-   (a) contacting a serum sample of said subject to the solid support    of the system,-   (b) adding sample of the detectably labelled probe of the system,    and-   (c) detecting probe bound antibody within said serum sample, wherein    a positive test is indicative of death of brain cells containing    neuromelanin and is characterised by an elevated level of the    indicator of release of neuromelanin compared to control values.

In a sixth aspect, the present invention relates to human NM or anantigenic fragment or analogue thereof when used to detect aneurodegenerative disease in a subject.

In a seventh aspect, the present invention relates to human NM or anantigenic fragment or analogue thereof when used to detect aneurodegenerative disease in a subject who is tested prior to having anyclinical symptoms of said neurodegenerative disease.

Typically, for the purposes of the first through seventh aspects of theinvention, the neurodegenerative disease is selected from the groupconsisting of idiopathic Parkinson's disease, and parkinsonism includingMultisystem Atrophy, Progressive Supranuclear palsy, Pick's disease,Corticobasal Degeneration and Dementia with Lewy Bodies. Morepreferably, the disease is idiopathic Parkinson's disease.

Definitions

As used herein the term Parkinson's disease (PD) is used to indicateboth idiopathic PD and the associated parkinsonian syndromescharacterised by dopaminergic degeneration within the SN. The associatedsyndromes known under the umbrella term of the parkinsonian syndromesinclude multisystem atrophy and progressive supranuclear palsy, andshare both some pathological and clinical features with the idiopathicParkinson's disease.

The term “analogue” as used herein with reference to neuromelanin meansa synthetic neuromelanin, or fragment thereof, which has thecharacteristic features of neuromelanin.

As used herein the term “neurodegenerative disease” refers to a diseasewhich is characterised by degeneration of neuromelanin-containingneuronal cells. Where the disease includes a preclinical phase the termneurodegenerative disease will be understood to include the preclinicalphase as well as, in all cases, to include the clinical phase of thedisease.

As used herein the term “treatment”, refers to any and all uses whichremedy a disease state or symptoms, or otherwise prevent, hinder,retard, or reverse the progression of disease or other undesirablesymptoms in any way whatsoever.

The term “therapeutically effective amount” as used herein, includeswithin its meaning a non-toxic but sufficient amount a compound orcomposition for use in the invention to provide the desired therapeuticeffect. The exact amount required will vary from subject to subjectdepending on factors such as the species being treated, the age andgeneral condition of the subject, the severity of the condition beingtreated, the particular agent being administered and the mode ofadministration and so forth. Thus, it is not possible to specify anexact “effective amount”. However, for any given case, an appropriate“effective amount” may be determined by one of ordinary skill in the artusing only routine experimentation.

In the context of this specification, the term “comprising” means“including principally, but not necessarily solely”. Furthermore,variations of the word “comprising”, such as “comprise” and “comprises”,have correspondingly varied meanings.

Any description of prior art documents herein, or statements hereinderived from or based on those documents, is not an admission that thedocuments or derived statements are part of the common general knowledgeof the relevant art in Australia or elsewhere.

In order that the present invention may be more clearly understoodpreferred forms will be described with reference to the followingexamples and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the progression of cellularpathology of Parkinson's disease.

FIG. 2 is a photomicrograph of extracellular neuromelanin (arrow heads)and intracellular neuromelanin (arrow) in a brain of a Parkinson'sdisease sufferer.

FIG. 3: Immune response in Parkinson's disease (PD), healthy age- andsex-matched control individuals without a family history of PD andindividuals suffering depression. The response in the PD group issignificantly higher than both the unrelated control group (p=0.005) andthe depression group (p=0.046).

FIG. 4 shows the results of the cross-reactivity of IgG responses to anumber of melanin analogues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with the general class ofneurodegenerative diseases, especially those generally classified underthe umbrella term of “Parkinson's disease”, including idiopathicParkinson's disease, and the parkinsonian diseases Multisystem Atrophy,Progressive Supranuclear palsy, Pick's disease, CorticobasalDegeneration and Dementia with Lewy Bodies.

Each of these diseases share the neuropathological characteristic thatthe NM-containing neurons of the substantia nigra degenerate and thusrelease NM. Other areas of the brain may also degenerate. Howeverbecause degeneration of the cells of the substantia nigra is typical ofeach of these diseases, and hence the release of NM is typical, each ofthe aforementioned neurodegenerative diseases may be detected bydetection of an indicator of release of NM.

Release of NM into the extracellular spaces, by degeneration ofdopaminergic cells of the SN, or other areas of the brain such as theLC, exposes the NM to the immune system of the individual. An immuneresponse is elicited and detection of that immune response provides anindication of release of NM through cell death.

In classical or idiopathic Parkinson's disease at least 65% of total SNdopaminergic neurons are lost prior to onset of the classical clinicalsymptoms of the disease. The triad of motor symptoms, tremor, rigidityand bradykinesia typify the onset of the clinical phase of the diseaseduring which the rate of loss of the remaining 35% of dopaminergic cellsis significantly slower than during the preclinical phase.

A humoral response to NM can be detected in a peripheral tissue, such asblood.

Accordingly, the present inventors describe an improved means fordiagnosing a neurodegenerative disease which may be used, for example,independently in diagnosis of a disease or to confirm a diagnosis madeon the basis of clinical symptoms. By providing a means which isindependent of clinical symptoms the present invention also makespossible the diagnosis of neurodegenerative disease, in particularParkinson's disease, before the onset of clinical symptoms. That is, byproviding a means of diagnosis of a pathological occurrence in aneurodegenerative disease rather than diagnosis based on clinicalsymptoms, the method makes possible diagnosis in the pre-clinical phaseof the neurodegenerative disease, such as Parkinson's Disease.

The method for detecting a neurodegenerative disease in a subjectinvolves testing the subject for an indicator of release of neuromelaninfrom cells in the brain. The method involves detection of an indirectindicator of the release of neuromelanin (NM) from cells in the brainwhich can be detected in subjects with clinical or preclinical stages ofneurodegenerative disease.

The indicator of release of NM may be circulating antibodies capable ofbinding to NM. The antibodies capable of binding to NM may also becapable of binding to an antigenic fragment of NM or an analogue of NM.Preferably, the analogue of NM is selected from the group consisting ofsynthetic dopamine melanin and synthetic noradrenaline melanin.

A positive test is indicative of death of brain cells containingneuromelanin and is characterised by an elevated level of the indicatorof release of neuromelanin compared to control values. Control valuesare determined by testing healthy individuals having no indications ofmotor dysfunction. The individuals may be age- and sex-matched for theindividuals being tested.

An elevated level of the indicator of release of NM reflects death ofdopaminergic neurons, which is associated with a neurodegenerativedisease in the subject.

In the process of detection a sample of a body fluid or tissue,containing an indicator of release of NM, is obtained from a testsubject. Preferably the tissue is blood. The skilled person willrecognise that the sample may be used in the performance of the methodimmediately or may be stored under suitable conditions until required.

Where the sample is a blood sample, serum may be isolated by standardmethods. The sera may be used immediately or stored under suitableconditions, for example at −80° C., until required.

A method of detection involves an antibody capable of reacting withneuromelanin, or an antigenic fragment or analogue thereof, and includesan antibody which is capable of specifically binding with theneuromelanin, or antigenic fragment or analogue thereof. An antibody iscapable of specifically binding with an antigen if it exhibits athreshold level of binding activity and/or it does not significantlycross-react with unrelated antigens. Antibodies herein specifically bindif they bind to said species with a binding affinity (Ka) of 10⁵ mol⁻¹or greater, typically 10⁶ mol⁻¹ or greater, preferably 10⁷ mol⁻¹ orgreater, more preferably 10⁸ mol⁻¹ or greater, and even more preferably10⁹ mol⁻¹ or greater. The binding affinity of an antibody can bedetermined, for example, by Scatchard analysis (G. Scatchard, Ann. NYAcad. Sci. 51, 660-672, 1949).

Detection of the antibodies can utilise any known means of detectingantibodies including radioimmunoassays, enzyme-linked immunosorbantassays, solid phase assays.

In radioimmunoassay, C14-labelled synthetic dopamine melanin issynthesized and immunoprecipitation methods used to detect limitingdilution concentrations of antibody to the antigenic determinant.

Preferably, the method utilises the enzyme-linked immunosorbant assay(ELISA) using NM isolated from the human brain or synthetic dopaminemelanin as the antigen. However, solid-phase immunoassay using syntheticneuromelanin immobilized on polystyrene and assayed by opticalagglutination techniques to determine limiting dilution provides analternate method of detection.

These methods are routinely employed in the detection ofantigen-specific antibody responses, and are well described in generalimmunology text books such as Immunology by Ivan Roitt, JonathanBrostoff and David Male (London: Mosby, c1998. 5th ed. andImmunobiology: Immune System in Health and Disease/Charles A. Janewayand Paul Travers. Oxford: Blackwell Sci. Pub., 1994), the contents ofwhich are herein incorporated by reference.

Further, in terms of treatment of a neurodegenerative disorder,therapeutic agents are often present in the form of pharmaceuticaland/or therapeutic formulations, that is, therapeutic agents presenttogether with a pharmaceutically acceptable carrier, adjuvant and/ordiluent.

Typically therapeutic agents include: antioxidants, iron chelators,nonamine oxidase inhibitors, apoptosis inhibitors, growth factors,dopamine receptor inhibitors, endogenous enzymes which protect againstoxidative damage such as glutathione, superoxide dismutase and catalase,inhibitors of excitatory damage, zonisamide, benzamide compounds, orethanesulfonyl-piperidine derivatives, or a combination thereof.

A preferred therapeutic agent is zonisamide, or an alkali metal saltthereof, and in relation to this, the disclosure of European PatentApplication No. EP 1 040 830 is incorporated herein by reference.

Another preferred therapeutic agent is selected from the benzamide groupof compounds, and in relation to this, the disclosure of U.S. Pat. No.6,140,369 is incorporated herein by reference. Typical therapeuticagents from the benzamide group of compounds are selected from the groupconsisting of: N-tert-butyl-4-acetamidobenzamide,N-iso-propyl-4-acetamidobenzamide, N-tert-amyl-4-acetamidobenzamide,N-tert-butyl-3-acetamidobenzamide,N-methylcyclopropyl-4-acetamidobenzamide,N-n-butyl-4-acetamidobenzamide, N-n-pentyl-2-acetamidobenzamide,N-tert-butyl-2-acetamidobenzamide, N-iso-butyl-4-acetamidobenzamide,N-n-propyl-4-acetamidobenzamide, N-n-propyl-4-acetamidobenzamide,N-1,2-dimethylpropyl-4-acetamidobenzamide,N-n-pentyl-4-acetamidobenzamide, N-2-methylbutyl-4-acetamidobenzamide,N-tert-butyl-2,3-diacetamidobenzamide,N-tert-amyl-2,4-diacetamidobenzamide,N-tert-butyl-2,5-diacetamidobenzamide,N-tert-butyl-2,6-diacetamidobenzamide, N-tert-butyl-3,4diacetamidobenzamide, N-tert-butyl-3,5-diacetamidobenzamide,N-iso-propyl-4-nitrobenzamide, N-tert-butyl-3-nitrobenzamide,N-tert-butyl-2-nitrobenzamide, N-n-butyl-4-nitrobenzamide,N-n-propyl-4-nitrobenzamide, N-tert-butyl-3,5-nitrobenzamide,N-tert-amyl-4-nitrobenzamide, N-1,2-dimethylpropyl-4-nitrobenzamide,N-n-butyl-3-nitrobenzamide, N-n-butyl-3,5-dinitrobenzamide,N-methylcylopropyl-4-nitrobenzamide, N-n-butyl-2-nitrobenzamide,N-n-pentyl-4-nitrobenzamide, N-2-methylbutyl-4-nitrobenzamide,N-n-pentyl-2-nitrobenzamide, N-1-methylpropyl-4-nitrobenzamide,N-tert-butyl-3-aminobenzamide, N-tert-butyl-4-aminobenzamide andN-methylcylopropyl-4-aminobenzamide, or a combination thereof.

A still further preferred therapeutic agent is selected from theethanesulfonyl-piperidine derivative group of compounds, and in relationto this, the disclosure of international (PCT) Publication No. WO00/75109 is incorporated herein by reference. Typical therapeutic agentsfrom the ethanesulfonyl-piperidine derivative group of compounds areselected from the group consisting of:4-[-2-(4-benzyl-piperidine-1-yl)-ethanesulfonyl]-phenol;4-[2-(4-p-tolyloxy-piperidine-1-yl)-ethanesulfonyl]-phenol; (−)-(3R,4R)-or(3S,4S)-4-benzyl-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-piperidin-3-ol;(+)-(3S,4S)- or(3R,4R)-4-benzyl-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-piperidin-3-ol;(3RS,4RS)-4-benzyl-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-piperidin-3-ol;(−)-(3R,4R)- or(3S,4S)-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-4-(4-methyl-benzyl)-piperidin-3-ol;(+)-(3R,4R)- or(3S,4S)-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-4-(4-methyl-benzyl)-piperidin-3-ol;and(3RS,4RS)-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-4-(4-methyl-benzyl)-piperidin-3-ol.

Typically, for medical use, salts of the therapeutic agents will bepharmaceutically acceptable salts; although other salts may be used inthe preparation of the compound or of the pharmaceutically acceptablesalt thereof. By pharmaceutically acceptable salt it is meant thosesalts which, within the scope of sound medical judgement, are suitablefor use in contact with the tissues of humans and lower animals withoutundue toxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art.

For instance, suitable pharmaceutically acceptable salts of compoundsuseful in the invention may be prepared by mixing a pharmaceuticallyacceptable acid such as hydrochloric acid, sulfuric acid,methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoicacid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaricacid, or citric acid.

For example, S. M. Berge et al. describe pharmaceutically acceptablesalts in detail in J. Pharmaceutical Sciences, 1977, 66:1-19. The saltscan be prepared in situ during the final isolation and purification ofthe therapeutic compound, or separately by reacting the free basefunction with a suitable organic acid. Representative acid additionsalts include acetate, adipate, alginate, ascorbate, asparate,benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate; nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like.

Also included within the scope of therapeutic agents are prodrugs.Typically, prodrugs will be functional derivatives of the therapeuticagents which are readily converted in vivo to the required (active)compound as active agents. Typical procedures for the selection andpreparation of prodrugs are known to those of skill in the art and aredescribed, for instance, in H. Bundgaard (Ed), Design of Prodrugs,Elsevier, 1985.

Single or multiple administrations of the therapeutic agents eitheralone or as a pharmaceutical composition can be carried out with doselevels and pattern being selected by the treating physician. Regardless,the therapeutic agents or pharmaceutical compositions should provide aquantity of the therapeutic agent sufficient to effectively treat thepatient.

One skilled in the art would be able, by routine experimentation, todetermine an effective, non-toxic amount of the therapeutic agent, orpharmaceutical composition containing the therapeutic agent, which wouldbe required to treat or prevent the disorders and diseases. Generally,an effective dosage is expected to be in the range of about 0.0001 mg toabout 1000 mg per kg body weight per 24 hours; typically, about 0.001 mgto about 750 mg per kg body weight per 24 hours; about 0.01 mg to about500 mg per kg body weight per 24 hours; about 0.1 mg to about 500 mg perkg body weight per 24hours; about 0.1 mg to about 250 mg per kg bodyweight per 24 hours; about 1.0 mg to about 250 mg per kg body weight per24 hours. More typically, an effective dose range is expected to be inthe range about 1.0 mg to about 200 mg per kg body weight per 24 hours;about 11.0 mg to about 100 mg per kg body weight per 24 hours; about 1.0mg to about 50 mg per kg body weight per 24 hours; about 11.0 mg toabout 25 mg per kg body weight per 24 hours; about 5.0 mg to about 50 mgper kg body weight per 24 hours; about 5.0 mg to about 20 mg per kg bodyweight per 24 hours; about 5.0 mg to about 15 mg per kg body weight per24 hours.

Alternatively, an effective dosage may be up to about 500 mg/m².Generally, an effective dosage is expected to be in the range of about25 to about 500 mg/m², preferably about 25 to about 350 mg/m², morepreferably about 25 to about 300 mg/m², still more preferably about 25to about 250 mg/m², even more preferably about 50 to about 250 mg/m²,and still even more preferably about 75 to about 150 mg/m².

Further, it will be apparent to one of ordinary skill in the art thatthe optimal quantity and spacing of individual dosages of a therapeuticagent will be determined by the nature and extent of the condition beingtreated, the form, route and site of administration, and the nature ofthe particular vertebrate being treated. Also, such optimum conditionscan be determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that theoptimal course of treatment, such as, the number of doses of thetherapeutic agent given per day for a defined number of days, can beascertained by those skilled in the art using conventional course oftreatment determination tests.

Whilst the therapeutic agent may be administered alone, it is generallypreferable that it be administered as a pharmaceuticalcomposition/formulation. In general pharmaceutical formulations may beprepared according to methods which are known to those of ordinary skillin the art and accordingly may include a pharmaceutically acceptablecarrier, diluent and/or adjuvant. The carriers, diluents and adjuvantsmust be “acceptable” in terms of being compatible with the otheringredients of the formulation, and not deleterious to the recipientthereof.

Examples of pharmaceutically and veterinarily acceptable carriers ordiluents are demineralised or distilled water; saline solution;vegetable based oils such as peanut oil, safflower oil, olive oil,cottonseed oil, maize oil, sesame oils such as peanut oil, saffloweroil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil orcoconut oil; silicone oils, including polysiloxanes, such as methylpolysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane;volatile silicones; mineral oils such as liquid paraffin, soft paraffinor squalane; cellulose derivatives such as methyl cellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose orhydroxypropylmethylcellulose; lower alkanols, for example ethanol oriso-propanol; lower aralkanols; lower polyalkylene glycols or loweralkylene glycols, for example polyethylene glycol, polypropylene glycol,ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin;fatty acid esters such as isopropyl palmitate, isopropyl myristate orethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth orgum acacia, and petroleum jelly. Typically, the carrier or carriers willform from 10% to 99.9% by weight of the compositions.

In a preferred form, the pharmaceutical compositions comprise aneffective amount of an active agent, together with a pharmaceuticallyacceptable carrier, diluent and/or adjuvant as shown in Example 2.

The pharmaceutical compositions may be administered by standard routes.In general, the compositions may be administered by the transdermal,intraperitoneal, intracranial, intracerebroventricular, intracerebral,oral, or parenteral (e.g., intravenous, intraspinal, subcutaneous orintramuscular) route. The compositions may be in the form of a capsulesuitable for oral ingestion, or in an aerosol form suitable foradministration by inhalation, such as by intranasal inhalation or oralinhalation.

For administration as an injectable solution or suspension, non-toxicparenterally acceptable diluents or carriers can include, Ringer'ssolution, isotonic saline, phosphate buffered saline, ethanol and1,2-propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvantsfor oral use include peanut oil, liquid paraffin, sodiumcarboxymethylcellulose, methylcellulose, sodium alginate, gum acacia,gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine andlecithin. In addition these oral formulations may contain suitableflavouring and colourings agents. When used in capsule form the capsulesmay be coated with compounds such as glyceryl monostearate or glyceryldistearate which delay disintegration of the capsule.

Adjuvants typically include emollients, emulsifiers, thickening agents,preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable inhuman and veterinary pharmaceutical practice, sweeteners, disintegratingagents, diluents, flavourings, coating agents, preservatives, lubricantsand/or time delay agents. Suitable binders include gum acacia, gelatine,corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose orpolyethylene glycol. Suitable sweeteners include sucrose, lactose,glucose, aspartame or saccharine. Suitable disintegrating agents includecorn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthangum, bentonite, alginic acid or agar. Suitable diluents include lactose,sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,calcium silicate or dicalcium phosphate. Suitable flavouring agentsinclude peppermint oil, oil of wintergreen, cherry, orange or raspberryflavouring. Suitable coating agents include polymers or copolymers ofacrylic acid and/or methacrylic acid and/or their esters, waxes, fattyalcohols, zein, shellac or gluten. Suitable preservatives include sodiumbenzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben,propyl paraben or sodium bisulfite. Suitable lubricants includemagnesium stearate, stearic acid, sodium oleate, sodium chloride ortalc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides, or mixtures thereof.

Suspensions for oral administration may further comprise dispersingagents and/or suspending agents. Suitable suspending agents includesodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginateor acetyl alcohol. Suitable dispersing agents include lecithin,polyoxyethylene esters of fatty acids such as stearic acid,polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate, andthe like.

The emulsions for oral administration may further comprise one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above, or natural gums such as guar gum, gumacacia or gum tragacanth.

The compositions for parenteral administration will commonly comprise asolution of an active agent or a cocktail thereof dissolved in anacceptable carrier, such as water, buffered water, 0.4% saline, and 0.3%glycine etc, wherein such solutions are sterile and relatively free ofparticulate matter.

Methods for preparing parenterally administrable compositions areapparent to those skilled in the art, and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa., hereby incorporated by referenceherein.

The pharmaceutical compositions may also be administered in the form ofliposomes. Liposomes are generally derived from phospholipids or otherlipid substances, and are formed by mono- or multi-lamellar hydratedliquid crystals that are dispersed in an aqueous medium. Any non-toxic,physiologically acceptable and metabolisable lipid capable of formingliposomes can be used. The formulations in liposome form may contain, inaddition to the therapeutic agent(s), stabilisers, preservatives,excipients and the like. The preferred lipids are the phospholipids andthe phosphatidylcholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art, and in relation to thisspecific reference is made to: Prescott, Ed., Methods in Cell Biology,Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., thecontents of which is incorporated herein by reference.

The invention will now be described in greater detail by reference tospecific Examples which should not be construed as in any way limitingthe scope of the invention.

EXAMPLES Example 1

Methods

Subjects

Fifty-two individuals with a clinical diagnosis of PD and fifty-onehealthy controls without any indications of motor dysfunction and sevenindividuals with a clinical diagnosis of severe depression wererecruited, with informed consent, in Sydney, Australia. The depressionsubjects represented a disease control group suffering a functional, butnot degenerative change, in central dopaminergic function. The age rangewas 43 to 88 years and did not differ between the PD (means age67.8±1.3), control (mean age 63.6±1.8), or depression subject (mean age64.5±6.5 p=0.44). A clinical diagnosis of PD was made if the total scoreobtained from Part III Motor Examination of the Unified ParkinsonDisease Rating Scale (Martinez-Martin, et al., 1994) was equal to fiveor above and the patient was levodopa responsive. No subject exhibitedany evidence of related motor disorders or of dementia. A further groupof twenty-seven patients with a clinical diagnosis of Parkinson'sdisease and thirty age- and sex-matched controls without any indicationsof motor dysfunction were recruited, with informed consent, in Bochum,Germany. The age range of the German subjects was 41 to 80 years and didnot differ between the PD (mean age 65.5±1.0) and control subjects (meanage 66.2±1.8 p=0.88. Neuropathological confirmation for the clinicaldiagnosis of PD was not available for any patient in this study.

Collection and Preparation of Serum Samples

Blood samples were collected with consent from each subject and the seraisolated by centrifugation. Sera were frozen at −80° C. until use.

Isolation of Human Neuromelanin

NM was isolated according to the method of Zecca and Swartz (1993) fromthe SN of 10 human subjects from Germany (age range: 41-64 years) withno history of neurological or neurodegenerative diseases. The SN weredissected from the brain within 36 hours of death on a cool plate (−10°C.) and pooled in a glass-Teflon homogeniser. The samples werehomogenised in 20 ml water and centrifuged at 12 000 g for 10 min. Theresulting pellets were washed twice with 50 mM phosphate buffer (pH7.4), then incubated in 50 mM Tris buffer (pH 7.4) containing 0.5mg.ml⁻¹ SDS at 37° C. for 3 h, followed by a further 3 h incubation withthe addition of 0.2 mg.ml⁻¹ proteinase K. The resulting pellets werepooled and consecutively washed with saline, water, methanol and hexane.The resulting dark pellet was incubated for three periods of 8 h each in150 mM EDTA (pH 7.4) before being washed twice with water and finallydried under vacuum.

Preparation of Synthetic Neuromelanins

Synthetic dopamine melanin (DAM) was prepared according to amodification of the method of Ben-Shachar et al. (1991). Two mM DA(Sigma, USA) was incubated in 50 mM Tris buffer (pH 8) containing 0.1 mMCuSO₄ for one month at room temperature. The resulting liquid wascentrifuged at 12 000 g for 15 min and the pellet was washed three timesin double distilled water at 4° C. The resulting melanin was finallylyophilised to a dry powder. Dopamine melanin was also synthesised using0.1 mM Fe₂(SO₄)₃ instead of copper (FeDAM). Other types of syntheticmelanins were produced using 2 mM noradrenaline (NAM) or serotonin(5HT-Mel) instead of dopamine. All chemicals used for synthetic melaninproduction were purchased from Sigma, Australia.

Enzyme-Linked Immunosorbant Assay

The ELISA was a modification of that reported by Rowe et al. (1998). Theantigen used was either human NM or synthetic melanin biotinylated usingEZ-Link Sulfo-NHS-LC-Biotin (Pierce, USA) according to themanufacturer's instructions and was prepared by sonification inphosphate-buffered saline to produce a suspension of fine, homogenousgranules at a concentration of 1 mg/ml. One hundred μL aliquots weredispensed into a 96-well Reacti-Bind NeutrAvidin with Superblock plate(Pierce, USA) sealed with an acetate sheet, protected from light andincubated at room temperature for 2 hours. The plate was washed threetimes with wash buffer consisting of 0.5% Tween 20 0.1% bovine serumAlbumin in Tris-buffered saline (pH 7.2 22° C.). 100 μL human seradiluted 1:50 in the wash buffer was added to each sextet of wells andincubated for 0.5 hr at room temperature. Following the primaryincubation, the plate was washed three times with wash buffer and 100 μLof horseradish peroxidase-conjugated goat antihuman IgG (Pierce,Rockford, USA) was added diluted to 1:10 000 in wash buffer andincubated for a further 0.5 hr. Following three further washes with washbuffer the bound signal was detected using citrated ABTS (0.3 mg/ml2-2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) activated with0.03% hydrogen peroxide in 0.1 M sodium citrate pH 4.5. Following anhour incubation at room temperature absorbance was determined at 405 nmusing a microplate reader (BioRad Benchmark, USA). Absorbance for eachsample was measured in six wells, corrected relative to wells containingno antigen and the mean absorbance calculated. Each plate containedpositive control samples consisting of pre- and anti-immune sera samplesraised against isolated human NM in the rabbit amplified by anti-rabbitIgG (Pierce, Rockford, USA) as the secondary antibody.

Synthetic dopamine melanin was replaced by isolated human NM prepared inthe same manner in some experiments.

Analysis

Absorbance results and effect of sex on absorbance were analysed usingthe Mann Whitney U test. Cross-reactivity was analysed using one-wayanalysis of variance while the effect of age on absorbance was analysedusing regression analysis.

Results

Results from the Sydney study are shown in FIG. 3 and demonstrate thatsera obtained from persons with a clinical diagnosis of PD (mean0.171±0.020) exhibits a significantly increased immune response on theELISA test compared with sera obtained from age- and sex-matched healthycontrol individuals (mean 0.108±0.011, p=0.005) and from individualssuffering from depression (mean 0.080±0.013, p=0.046) using syntheticDAM as the antigen. A similar response was seen in the German studywhere the immune response of the PD patients (mean 0.109±0.007) wassignificantly higher (p=0.001) than the controls (mean 0.079±0.003).

The specificity of the immune response was determined by testing thecross-reactivity of the sera for various substrates. The significantlydifferent absorbance response stimulated by exposure of parkinsoniancompared with control sera to DAM was also exhibited when FeDAM or NAMwere used as the antigen (both p<0.001). In contrast, no difference inabsorbance was recorded when 5HT-Mel was used as the assay antigen(p=0.64, FIG. 3). Sera obtained from 48 healthy individuals aged from 21to 92 years demonstrated the absorbance response was not significantlyaffected by the age (p=0.10) or sex (p=0.12) of the serum donor.

Discussion

The present invention provides a new and inexpensive method to identifyan immune response in PD patients to DAM and isolated human NM. Thisresponse indicates the release of NM from central dopaminergic neuronsand thus the death of neurons within the central dopaminergic system.Such a test could confirm a clinical diagnosis of PD or indicate thepresence of preclinical PD in cases where clinical symptomology are notyet present. The results demonstrate that an enhanced IgG response isassociated with a clinical diagnosis of PD and that the response isspecific for catecholaminergic-based melanins. A slightly enhanced IgGresponse in PD patients was also identified in assays in which DAM wasreplaced by NAM as the antigen. This response is consistent with theloss of the noradrenergic neurons of the locus ceruleus in PD as theseneurons also form a melanin based upon noradrenaline, rather thandopamine, which is thought to be structurally similar to NM (Double etal., 1999). It is unknown whether the increased response to NAM resultedfrom a specific response from noradrenaline neuromelanin-stimulated IgGor a combined response to DAM and NAM. The locus coruleus, contains,however, only 40,000 pigmented cells compared with the approximately400,000 pigmented cells within the SN, so that the magnitude of aspecific immune response to the loss of noradrenaline neuromelanin islikely to be significantly less than that induced by the simultaneousdeath of dopaminergic cells of the SN.

Currently, no other method can reliably identify death of centraldopaminergic cells in vivo. The identification of the release of NM fromthese cells by the method of the present invention represents a specificand sensitive technique to determine neurodegeneration of centraldopaminergic systems.

The present inventors discovered that the detection of the release of NMoccurring at cell death would form a specific marker for the death ofthese melanised cells and thus for the disorders characterised by thedeath of these cells, even prior to the onset of symptoms. The methoddisclosed herein provides a means of detecting a humoral IgG response tothe release of NM from dying cells which utilises the enzyme-linkedimmunosorbant assay (ELISA) and either NM isolated from the human brainor synthetic dopamine melanin as the antigen. This assay demonstratedthat a humoral response to NM can be detected in a peripheral tissue(blood) and that this response is significantly enhanced in patientssuffering from clinical symptoms of PD.

Another element which also may stimulate an immune response in PD isLewy Bodies (LB). LB are pathological inclusions which form inside thedopaminergic-neurons in PD and, like NM, are released upon death ofthese cells. LB contain modified forms of some proteins (such assynuclein and neurofilaments) which aggregate into round inclusionbodies. Detection of antibodies to released LB may also be a means of isdetecting a neurodegenerative disease in a subject in the clinical orpre-clinical phase of said neurodegenerative disease.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

Example 2 Pharmaceutical Formulations

The therapeutic agent(s) for use in the present invention may beadministered alone, although it is preferable that they be administeredas a pharmaceutical formulation. The active ingredient may comprise,from 0.001% to 10% by weight, and more typically from 1% to 5% by weightof the formulation, although it may comprise as much as 10% by weight.

Specific preferred pharmaceutical compositions are outlined below.However, the following are to be construed as merely illustrativeexamples of formulations and not as a limitation of the scope of thepresent invention in any way.

Example 2(a) Composition for Inhalation Administration

For an aerosol container with a capacity of 20-30 ml: a mixture of 10 mgof zonisamide with 0.5-0.8% by weight of a lubricating agent, such aspolysorbate 85 or oleic acid, is dispersed in a propellant, such asfreon, and put into an appropriate aerosol container for eitherintranasal or oral inhalation administration.

Example 2(b) Composition for Parenteral Administration

A pharmaceutical composition for intramuscular injection could beprepared to contain 1 mL sterile buffered water, and 1 mg of zonisamide.

Similarly, a pharmaceutical composition for intravenous infusion maycomprise 250 ml of sterile Ringer's solution, and 5 mg of zonisamide.

Example 2(c) Capsule Composition

A pharmaceutical composition in the form of a capsule may be prepared byfilling a standard two-piece hard gelatin capsule with 50 mg ofzonisamide, in powdered form, 100 mg of lactose, 35 mg of talc and 10 mgof magnesium stearate.

Example 2(d) Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by injection may be prepared by mixing 1% by weight ofzonisamide in 10% by volume propylene glycol and water. The solution issterilised by filtration.

REFERENCES

-   Ben-Shachar, D., Riederer, P. and Youdim, M. B. H., “Iron-melanin    interaction and lipid peroxidation: implications for Parkinson's    disease”, J. Neurochem., 57 (1991) 1609-1614.-   Double, K., Riederer, P. and Gerlach, M., “Significance of    neuromelanin for neurodegeneration in Parkinson's disease”, Drug    News Dev, 12 (1999) 333-340.-   Fearnley, J. and Lees, A., “Ageing and Parkinson's disease:    substantia nigra regional selectivity”, Brain, 114 (1991) 2283-2301.-   Halliday, G., McRitchie, D., Cartwright, H., Pamphlett, R., Hely, M.    and Morris, J., “Midbrain neuropathology in idiopathic Parkinson's    disease and diffuse Lewy body disease”, J Clin. Neurosci., 3 (1996)    52-60.-   Martinez-Martin, P., Gil-Nagel, A., Balseiro Gomez, J., Morlan,    Gracia, L., Martinez-Sarries, J., Bermejo, F. and The Cooperative    Multicentre Group. “Unified Parkinson's disease Rating Scale    Characteristics and Structure”. Mov. Disorders 9 (1994) 76-83.-   Morrish, P., Rakshi, J., Bailey, D., Sawle, G. and Brooks, D.,    “Measuring the rate of progression and estimating the preclinical    period of Parkinson's disease with (18F)dopa PET”., J Neurol    Neurosurg Psychiatry, 64 (1998) 314-319.-   Rowe, D., Le, W., Smith, G. and Appel, S., “Antibodies from patients    with Parkinson's disease react with protein modified by dopamine    oxidation”, J. Neurosci. Res., 53 (1998) 551-558.-   Zecca, L. and Swartz, H. M., “Total and paramagnetic metals in human    substantia nigra and its neuromelanin”, J. Neural Trans. Park. Dis.    Sect., 5 (1993) 203-213.

1. A method for detecting a neurodegenerative disease in a subject, saidmethod comprising testing the subject for an indicator of release ofneuromelanin from cells in the brain, wherein a positive test isindicative of death of brain cells containing neuromelanin and ischaracterised by an elevated level of the indicator of release ofneuromelanin compared to control values.
 2. The method of claim 1,wherein the subject is tested prior to having any clinical symptoms ofsaid neurodegenerative disease.
 3. The method of claim 1, wherein saidcell death is associated with the neurodegenerative disease in thesubject.
 4. The method of claim 1, wherein the neurodegenerative diseasehas clinical symptoms selected from the group consisting of: tremor,rigidity, bradykinesia and slowness of movement.
 5. The method of claim1, wherein the indicator is an immune response in the form ofcirculating antibodies to neuromelanin, or analogues thereof, in saidsubject.
 6. The method of claim 5, wherein the detection method employsthe detection of antibodies capable of reacting to neuromelanin, or anantigenic fragment or analogue thereof, present in said subject.
 7. Themethod of claim 6, wherein the analogue of neuromelanin is selected fromthe group consisting of synthetic dopamine melanin and syntheticnoradrenaline melanin.
 8. The method of, claim 1 wherein said methodcomprises: (a) isolating sera from a blood sample from said subject; (b)incubating said sera with an antigen selected from human NM or syntheticdopamine melanin under conditions suitable for antibody-antigen binding;and (c) detecting bound antibody.
 9. A method of treatment of aneurodegenerative disease in a subject, said method comprising: (a)screening subjects to detect a neurodegenerative disease using themethod of claim 1, and (b) initiating treatment for subjects testedpositive for the indicator of release of neuromelanin from cells in thebrain.
 10. The method of claim 9, wherein said treatment comprisesadministering a therapeutically effective amount of at least one of thefollowing: antioxidants, iron chelators, nonamine oxidase inhibitors,apoptosis inhibitors, growth factors, dopamine receptor inhibitors,zonisamide, benzamide compounds, ethanesulfonyl-piperidine derivatives,endogenous enzymes which protect against oxidative damage or inhibitorsof excitatory damage, or a combination thereof.
 11. The method of claim10, wherein said benzamide compound is selected from the groupconsisting of: N-tert-butyl-4-acetamidobenzamide,N-iso-propyl-4-acetamidobenzamide, N-tert-amyl-4-acetamidobenzamide,N-tert-butyl-3-acetamidobenzamide,N-methylcyclopropyl-4-acetamidobenzamide,N-n-butyl-4-acetamidobenzamide, N-n-pentyl-2-acetamidobenzamide,N-tert-butyl-2-acetamidobenzamide, N-iso-butyl-4-acetamidobenzamide,N-n-propyl-4-acetamidobenzamide, N-n-propyl-4-acetamidobenzamide,N-1,2-dimethylpropyl-4-acetamidobenzamide,N-n-pentyl-4-acetamidobenzamide, N-2-methylbutyl-4-acetamidobenzamide,N-tert-butyl-2,3-diacetamidobenzamide,N-tert-amyl-2,4-diacetamidobenzamide,N-tert-butyl-2,5-diacetamidobenzamide,N-tert-butyl-2,6-diacetamidobenzamide,N-tert-butyl-3,4-diacetamidobenzamide,N-tert-butyl-3,5-diacetamidobenzamide, N-iso-propyl-4-nitrobenzamide,N-tert-butyl-3-nitrobenzamide, N-tert-butyl-2-nitrobenzamide,N-n-butyl-4-nitrobenzamide, N-n-propyl-4-nitrobenzamide,N-tert-butyl-3,5-nitrobenzamide, N-tert-amyl-4-nitrobenzamide,N-1,2-dimethylpropyl-4-nitrobenzamide, N-n-butyl-3-nitrobenzamide,N-n-butyl-3,5-dinitrobenzamide, N-methylcylopropyl-4-nitrobenzamide,N-n-butyl-2-nitrobenzamide, N-n-pentyl-4-nitrobenzamide,N-2-methylbutyl-4-nitrobenzamide, N-n-pentyl-2-nitrobenzamide,N-1-methylpropyl-4-nitrobenzamide, N-tert-butyl-3-aminobenzamide,N-tert-butyl-4-aminobenzamide and N-methylcylopropyl-4-aminobenzamide,or a combination thereof.
 12. The method of claim 10, wherein saidethanesulfonyl-piperidine derivative is selected from the groupconsisting of 4-[-2-(4-benzyl-piperidine-1-yl)-ethanesulfonyl]-phenol;4-[2-(4-p-tolyloxy-piperidine-1-yl)-ethanesulfonyl]-phenol; (−)-(3R,4R)-or(3S,4S)-4-benzyl-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-piperidin-3-ol;(+)-(3S,4S)- or(3R,4R)-4-benzyl-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-piperidin-3-ol;(3RS,4RS)-4-benzyl-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-piperidin-3-ol;(−)-(3R,4R)- or(3S,4S)-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-4-(4-methyl-benzyl)-piperidin-3-ol;(+)-(3R,4R)- or(3S,4S)-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-4-(4-methyl-benzyl)-piperidin-3-ol;and(3RS,4RS)-1-[2-(4-hydroxy-benzenesulfonyl)-ethyl]-4-(4-methyl-benzyl)-piperidin-3-ol,or a combination thereof.
 13. The method of claim 10, wherein saidendogenous enzymes which protect against oxidative damage are selectedfrom the group consisting of: glutathione, superoxide dismutase andcatalase, or a combination thereof.
 14. The method of claim 10, whereinsaid treatment comprises administering a therapeutically effectiveamount of zonisamide.
 15. The method of claim 1, wherein theneurodegenerative disease is selected from the group consisting ofidiopathic Parkinson's disease, and parkinsonism including MultisystemAtrophy, Progressive Supranuclear palsy, Pick's disease, CorticobasalDegeneration and Dementia with Lewy Bodies.
 16. The method of claim 15,wherein the neurodegenerative disease is idiopathic Parkinson's disease.17. A system for the detection of a neurodegenerative disease in asubject, said system comprising: (a) means for capturing an indicator ofrelease of neuromelanin from cells in the brain of a subject; and (b)means for detecting the captured indicator of release of neuromelaninfrom cells in the brain.
 18. The system of claim 17, wherein saidindicator of release of neuromelanin is circulating antibodies toneuromelanin, or analogues thereof, in said subject.
 19. The system ofclaim 17, wherein said means for capturing the indicator of release ofneuromelanin is neuromelanin or an antigenic fragment or analoguethereof immobilized to a solid surface.
 20. The system of claim 17,wherein said system comprises neuromelanin or an antigenic fragment oran analogue thereof, bound to a solid support and a source of adetectably labelled probe.
 21. The system of claim 20, wherein saidprobe is a labelled antihuman IgG.
 22. The system of claim 20, whereinsaid probe is horseradish peroxidase-conjugated goat antihuman IgG. 23.The system of claim 17, wherein said system is an ELISA-based system.24. The system of claim 17, wherein said system is provided in kit form.25. The system of claim 17, wherein the neurodegenerative disease isselected from the group consisting of idiopathic Parkinson's disease,and parkinsonism including Multisystem Atrophy, Progressive Supranuclearpalsy, Pick's disease, Corticobasal Degeneration and Dementia with LewyBodies.
 26. The system claim 24, wherein the neurodegenerative diseaseis idiopathic Parkinson's disease.
 27. A method for detecting aneurodegenerative disease in a subject comprising testing the subjectfor an indicator of release of neuromelanin from cells in the brain,said method comprising (a) contacting a serum sample of said subject toa detection system comprising neuromelanin, or an antigenic fragment oran analoque thereof, bound to a solid support, (b) adding sample of thedetectably labelled probe that binds the indicator of release ofneuromelanin from cells in the brain, and (c) detecting probe boundantibody within said serum sample, wherein a positive test is indicativeof death of brain cells containing neuromelanin and is characterised byan elevated level of the indicator of release of neuromelanin comparedto control values.
 28. The method of claim 27, wherein theneurodegenerative disease is selected from the group consisting ofidiopathic Parkinson's disease, and parkinsonism including MultisystemAtrophy, Progressive Supranuclear palsy, Pick's disease, CorticobasalDegeneration and Dementia with Lewy Bodies.
 29. The method of claim 27,wherein the neurodegenerative disease is idiopathic Parkinson's disease.30. The method of claim 27, wherein said subject is tested for thepresence of said neurodegenerative disease prior to having any clinicalsymptoms of said neurodegenerative disease.